Method for preparing alpha-amylase

ABSTRACT

A METHOD FOR PREPARING A-AMYLASE BY DEPTH CULTIVATION OF ASPERGILLUS ORYZAE ON A WATER NUTRIENT MEDIUM CONTAINING THE FOLLOWING INGREDIENT: STARCH, NANO3, MGSO4, KCL, FESO4, KH2PO4, MG(NO3)2, MG(H2PO4)2 AND 20 PERCENT BY VOLUME OF 20 PERCENT EXTRACT OF MALT SPROUTS. THE CULTURE FILTRATE IS DIALYZED WITH PHOSPHATE BUFFER AT PH 6.5-7.5. FORM THE DIALYZATE, THE A-AMYLASE IS ADSORBED ON DIETHYLAMINE ETHYLCELLULOSE AND THEN ELUTED WITH A PHOSPHATE BUFFER (FROM 0.04 TO 0.12M) AT PH 6.5-7.5 CONTAINING CACL2 (0.0003-0.001M).

United States Patent METHOD FOR PREPARING (Z-AMYLASE Georgf-lvanovichKvesitadze, 'uli'tsa Abasheli 7a, and t GeorgyNikolaevichFKokonaShViIi,ulitsa Perovskoi 4, -==b'oth of Tbilisi, U.S.S.R., and Raisa VasilievnaFenixrova, 'ulitsaNovoslobodskaya.67/ 69, kv. 167, Moscow,

U.S.S.R. v I No Drawing. Continuation of abandoned application Ser.v151,0...189,032, Oct 13,1971. This application Apr. 2,

197. eri -3 6, 8 "Claims priority, application U.S.S.R., Oct. 20, 1970,

J Int. Cl. (212d 13/10 Cl. 195 --66R 1 This' is a continuation ofapplication Ser. No. 189,032, filed Oct. 13,1971, and now abandoned.

This invention relates to the preparation of enzymes, and moreparticularly itrelates to a method for the prepara'tion of tit-amylase.

' u-Am ylase'has 'beerffound in'animals (in saliva, pancreas"), inplants (malt), in moldsand bacteria. All these enzymes are a- 1,4glucane 4 glucohydralases (3,2,1,1). They'cat alyze the'hydrolysis ofstarch, glycogen and related a-l,4 glucan es. Said enzymes however,ditfer by their molecular weight, thermal stability, optimum pH andother characteristics." Very widely used are various preparationso ffungal "bf-amylase; namely in distillery, bakery, and beer'manufact'u're to hydrolyse naturaloligoandpolysaccharides"ttifermentable sugars. Medical prep arations aremanufactured on the basis of highly purified b t-amylase obtained froni'fungus Aspergillus oryzae. 'a' Amyls ases isolated "from varioussources differ by heat stability. Most sensitive toward temperature isuamylase isolated from Aspe'rgillusfmalt amylase is more stable.and"the. -greatest.stability to temperature is in aarnylase iisolatedfromibacteriar The enzymes differ also with respect to optimum pH.Fungal a-amylase has an optimum pH of 4.7, salivery and bacteriala-amylas'eha'veopti'mum pHs of 7.0 and 7.2, due to which the fungal ra-amylase is more suitable for the manufacture of medicinal preparationsused in treating gastrointestinal diseases.

Out of many known methods for preparing tit-amylase, most suitable forindustrial application is the method developed by Japanese investigators(Anabori et al., 1. Biochem. 1954, 41, 577) and modified in the U.S.S.R.(R. V. Feniksova, and G. A. Molodova, Moscow, 1961, J. Microbiology,vol. XXX, issue 4, p. 607).

The method comprises the following operations.

Technical preparation of the enzymes includes isolation by precipitationwith organic solvents (ethyl alcohol, as a rule) from an aqueous extractof the culture of Aspergz'llus oryzae grown superficially on wheat bran.

The precipitated preparation is dissolved in water, and

passed through a filter. To the filtrate is added an equal volume ofcalcium acetate solution (0.25M) and the pH of the solution is adjustedto 7.0. The precipitate is sep- "ice arated on a centrifuge, thefiltrate is treated with ammonium sulphate, the obtained precipitate isdissolved in water and dialyzed. a-Amylase is precipitated from thedialyzate with 1 percent solution of rivanol, and centrifuged. Theobtained precipitate is dissolved in acetate butler at pH 5.5. Thesolution is decolorized by shaking With Japanese clay and precipitatedwith a solution of acetone in the cold at a concentration of 55 percent.The centrifuged precipitate is dissolved with chilled solution of amixture of calcium acetate and sodium acetate having a concentration of0.02M. Acetone is added gradually into the solution of ot-amylase in thecold until turbidity develops. The solution is then allowed to stand incold until a-amylase crystals appear. The yield of the thus preparedint-amylase is 20-30 percent of theory.

The disadvantage of the described method is low yield of tat-amylase,which is only 25-30 percent and a great number of labour consumingoperations. It should be noted also that the final product is notcompletely homogeneous with respect to proteins and in electrophoresison polyacrylamide gel it produces three protein lines.

Known also is the method for preparing tit-amylase by depth cultivatingAspergillus oryzae on a water nutrient medium having the followingcomposition in g./litre: starch 90, NaNO 12, MgSO', 0.65, KH PO 1.3, KCl0.5, FeSO 0.02 and 200 ml. of 20 percent extract of malt sprouts for 72hours at a temperature of 30 C. (See Journal of Applied Biochemistry andMicrobiology, vol. V, issue 2 U.S.S.R. Academy of Science, 1969, pp.141- 146). Cultivation of mold Aspergi'lus oryzae on said medium inconditions of optimum aeration allows the production of a culture fluidwith a highrlevel biosynthesis of aamylase.

In this connection the object of this invention is to improve thecomposition of the culture medium by intro ducing new ingredients whichpromote biosynthesis of aamylase.

Another object of the invention is improvement of the process ofpurification of iii-amylase.

Still another object of the invention is to work out a method forpreparation of completely homogeneous (X- amylase with respect toprotein, and finally the object of the invention is to develop a methodfor preparation of a-amylase that includes the minimum possibleoperations and is suitable for industrial realization.

This and other objects of the invention have been realized in the methodfor preparation of a-amylase by depth cultivation of Aspergillus oryzaewith aeration on an aqueous nutrient medium containing starch, NaNO MgSOKH PO KCl, Fe'SO and malt sprout extract.

According to the invention said mold is cultivated on a nutrient mediumcontaining the following components in g./litre: starch from 50 to 90,NaNO from 8 to 15, MgSO from 0.4 to 1.5, KH PO from 0.2 to 1.2, KCl 0.5,FeSO from 0.001 to 0.08, Mg(NO from 0.2 to 0.8, Mg(H PO from 0.1 to 0.7and 20 percent extract of malt sprouts 20 percent by volume.

Introduction of Mg(NO- and Mg(H PO into the nutrient medium promotesintense secretion of a-amylase from mycelium of the fungus into theculture fluid.

Cultivation is carried out at a temperature from 28 to 32 C. for 65-80hours. The obtained culture fluid is separated on a filter fromMycelium; the filtrate which is a-amylase is dialyzed with a phosphatebuffer having a concentration from 0.0003 to 0.003 M at pH 6.57.5. Thephosphate buffer is a mixture of the salts KH PO and Na HPO During thedialysis, the solution of iii-amylase is freed from a part of pigments,remnants of mineral salts and other low-molecular metabolites. Theobtained dialysate, freed from the said substances, is passed throughdiethylamine ethylcellulose upon which the a-amylase and other proteinsare completely adsorbed.

In order to isolate a-amylase and to separate it from the accompanyingproteins, two-step elution is used, wherein tat-amylase is eluted withthe said phosphate buffer. First the adsorbing material is treated withthe phosphate buffer (0.04-0.09M) at pH 6.5-7.5 to elute theaccompanying proteins. Then the adsorbing material is processed with thesaid phosphate buffer (0.1-0.12 M) at pH 6.5-7.5 containing CaCl atconcentration from 0.0003 to 0.001 M, as a result of which a fraction ofa-amylase free from other proteins is obtained. The adherence to thisorder of the elution is a very important factor which ensures preparation of u-amylase which is homogeneous with respect to proteins. Theyield of the product is as high as 100 percent. No other process knownin the art has ever ensured yields of that order.

Thus, the proposed method for preparation of a-amylase reduces thenumber of process steps several times compared with the processes knownin the prior art.

After lyophilization a-amylase prepared by the proposed method can bestored at C. for three years, the activity of the preparation beinglowered not more than 10 percent.

For a better understanding of the present invention by those skilled inthe art the following example of an embodiment of the proposed inventionis given by way of illustration.

Example Conidia of a ten-day culture of Aspergillus oryzae 3-9-15 grownin a test tube on a 7 percent aqueous solution of beer must containing 2percent of agar-agar, are washed with sterile tap water and transferredin -ml. portions into fiasks on a shaker containing nutrient medium ofthe following composition in g./litre: starch 60, NaNO 9.0, MgSO.; 1.0,KH PO 1, KCl 0.5, FeSO 0.03, Mg(NO 0.2, Mg(H PO 0.1 and percent extractof malt sprouts 10 percent by volume. The seeding culture is grown for30 hours on a shaker reciprocating at a speed of 1-80 r.p.m., at atemperature of 30 C. Then the seeding culture (0.5 percent) istransferred in sterile conditions into a fermentation tank of 1000-litrecapacity holding 500 litres of nutrient medium containing, in g./ litre:starch 80, NaNO 12, KH PO 1, MgSO.; 1, KCl 0.5, FeSO 0.03, Mg(-NO 0.8,Mg('H PO 0.5 and 20 percent extract of malt sprouts 20 percent byvolume.

The fermentation is continued for 72 hours at a temperature of 30 C. Thefermentation tanks are equipped with spargers through which the air isbubbled, and stirrers. The aeration rate during the first. 24 hours is250 litres per minute, and afterwards the aeration is intensified to 500litres per minute. To extinguish the foam, sperm oil is used. (Oleinicacid and other antifoam agents can also be used). 0n termination of thefermentation process, the mycelium is separated on a filter or aseparator.

The activity of a-amylase in the culture filtrate is 20 units/ml. Thefiltrate is dialyzed with 0.003M phosphate buffer at pH 7.15 for 12hours. The obtained dialyzate is divided into several equal portionscontaining 1000 mg. of protein each and each having the activity ofu-amylase of 10,000 units. Each portion ofthe dialyzate is passedthrough an individual column packed with 10 got diethylamineethylcellulose. The diameter of the column is 35 mm., the height 250 mm.During this operation aamylase and the other proteins are absorbed onthe column P I Next the a-amylase is eluted from the sorbent. Theprocess is carried out in two steps. The first elution is effected witha 0.06M phosphate bufier at pH7.15. During this operation only theaccompanying proteins are eluted whereas the a-amylase remains on thecolumn packing.

a-Amylase is eluted during the second step when a 0.11 M phosphatebuffer at pH 7.15 containing 0001M solution of CaCl is passed throughthe column. Thesaid buffer completely elutes the fraction containinga-amylase. The yield of a-amylaseis 300 mg., having a total activity of10;000 units, that is the yield of a-amylase after elution is 100percent. Dialysis, adsorption and elution of proteins is carried out ata temperature of 5+8 C.

The protein homogeneity of the thus obtained atamylase was as follows:in ultracentrifuging one protein peak was obtained and inelectrophoresis on-polyacrylamide gel there was one protein line. Duringa repeated adsorption of the a-amylase fraction on diethylamineethylcellulose and subsequent elution, no u-amylase' fraction inactivewith respect to a-amylase was separated.

The specific activity of u-amylase obtained according to the proposedmethod was almosttwice as great compared with the activity oftat-amylase obtained 'by the known method.

What is claimed is:

1. A method for preparing a-amylase which comprises depth cultivating ofmold Aspergillus oryzae with aeration on a water nutrient medium of thefollowing composition, in g. per litre: starch from 50 to 90, NaNO from8 to 15, MgSO; from 0.4 to 1.5, KH PO from 0.2 to 1.2, KCl 0.5, FeSOfrom 0.001 -to 0.08, Mg(NO from 0.2 to 0.8, Mg(H PO from 0.1 to 0.7 and20 percent extract of malt sprouts 20 percent by volume, filtering theculture fluid, dialyzing the obtained culture filtrate with 0.0003 to0.003 M phosphate buffer at pH 6.5-7.5, passing the dialyzate throughdiethylamine ethylcellulose upon which tat-amylase is adsorbed, elutingproteins inactive with respect to a-amylase with 0.04-0.09 M phosphatebuffer at pH 6.57.5 and eluting tat-amylase with 0.10.12.M phosphatebuffer at pH 6.57.5 containing CaCl in concentration from 0.0003 to0.001 M. 1

2. The method of claim 1 wherein cultivation is carried out at atemperature of from 28 to 32 C. for to hours. A l

References Cited Journal of Applied Biochemistry and Microbiology, vol.V, issue Z, U.S.S:R. Academy :of Science, 1969, pp.

LIONEL M. SHAP'IRQ Primary Examiner

